Genome Editing Strategies Research Articles

Restored Collagen VI Microfilaments Network in the Extracellular Matrix of CRISPR-Edited Ullrich Congenital Muscular Dystrophy Fibroblasts

Collagen VI is an essential component of the extracellular matrix (ECM) composed by α1, α2 and α3 chains and encoded by COL6A1, COL6A2 and COL6A3 genes. Dominant negative pathogenic variants in COL6A genes result in defects in collagen VI protein and are implicated in the pathogenesis of muscular diseases, including Ullrich congenital muscular dystrophy (UCMD). Here, we designed a CRISPR genome editing strategy to tackle a dominant heterozygous deletion c.824_838del in exon 9 of the COL6A1 gene, causing a lack of secreted collagen VI in a patient’s dermal fibroblasts. The evaluation of efficiency and specificity of gene editing in treating patient’s fibroblasts revealed the 32% efficiency of editing the mutated allele but negligible editing of the wild-type allele. CRISPR-treated UCMD skin fibroblasts rescued the secretion of collagen VI in the ECM, which restored the ultrastructure of the collagen VI microfibril network. By using normal melanocytes as surrogates of muscle cells, we found that collagen VI secreted by the corrected patient’s skin fibroblasts recovered the anchorage to the cell surface, pointing to a functional improvement of the protein properties. These results support the application of the CRISPR editing approach to knock out COL6A1 mutated alleles and rescue the UCMD phenotype in patient-derived fibroblasts.

Current Updates of CRISPR/Cas System and Anti-CRISPR Proteins: Innovative Applications to Improve the Genome Editing Strategies.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated sequence (CRISPR/Cas) system is a cutting-edge genome-editing tool employed to explore the functions of normal and disease-related genes. The CRISPR/Cas system has a remarkable diversity in the composition and architecture of genomic loci and Cas protein sequences. Owing to its excellent efficiency and specificity, this system adds an outstanding dimension to biomedical research on genetic manipulation of eukaryotic cells. However, safe, efficient, and specific delivery of this system to target cells and tissues and their off-target effects are considered critical bottlenecks for the therapeutic applications. Recently discovered anti-CRISPR proteins (Acr) play a significant role in limiting the effects of this system. Acrs are relatively small proteins that are highly specific to CRISPR variants and exhibit remarkable structural diversity. The in silico approaches, crystallography, and cryo-electron microscopy play significant roles in elucidating the mechanisms of action of Acrs. Acrs block the CRISPR/Cas system mainly by employing four mechanisms: CRISPR/Cas complex assembly interruption, target-binding interference, target cleavage prevention, and degradation of cyclic oligonucleotide signaling molecules. Engineered CRISPR/Cas systems are frequently used in gene therapy, diagnostics, and functional genomics. Understanding the molecular mechanisms underlying Acr action may help in the safe and effective use of CRISPR/Cas tools for genetic modification, particularly in the context of medicine. Thus, attempts to regulate prokaryotic CRISPR/Cas surveillance complexes will advance the development of antimicrobial drugs and treatment of human diseases. In this review, recent updates on CRISPR/Cas systems, especially CRISPR/Cas9 and Acrs, and their novel mechanistic insights are elaborated. In addition, the role of Acrs in the novel applications of CRISPP/Cas biotechnology for precise genome editing and other applications is discussed.

Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34+ cell therapy to induce fetal hemoglobin for sickle cell disease

Sickle cell disease (SCD) is a common, severe genetic blood disorder. Current pharmacotherapies are partially effective and allogeneic hematopoietic stem cell transplantation (HSCT) is associated with immune toxicities. Genome editing of patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers an alternative potentially curative approach to treat SCD. Although the FDA released guidelines for evaluating genome editing risks, it remains unclear how best to approach pre-clinical assessment of genome-edited cell products. Here we describe rigorous pre-clinical development of a therapeutic γ-globin gene promoter editing strategy that supported an investigational new drug (IND) application cleared by the FDA. We compared γ-globin promoter and BCL11A enhancer targets, identified a potent HbF-inducing lead candidate, and tested our approach in mobilized CD34+ HSPCs from SCD patients. We observed efficient editing, HbF induction to predicted therapeutic levels, and reduced sickling. With single-cell analyses, we defined the heterogeneity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased off-target discovery followed by targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new ex vivo HSC genome editing strategies towards clinical trials for treating SCD and other blood disorders.

Gene Therapy for Retinitis Pigmentosa: Current Challenges and New Progress.

Retinitis pigmentosa (RP) poses a significant threat to eye health worldwide, with prevalence rates of 1 in 5000 worldwide. This genetically diverse retinopathy is characterized by the loss of photoreceptor cells and atrophy of the retinal pigment epithelium. Despite the involvement of more than 3000 mutations across approximately 90 genes in its onset, finding an effective treatment has been challenging for a considerable time. However, advancements in scientific research, especially in gene therapy, are significantly expanding treatment options for this most prevalent inherited eye disease, with the discovery of new compounds, gene-editing techniques, and gene loci offering hope for more effective treatments. Gene therapy, a promising technology, utilizes viral or non-viral vectors to correct genetic defects by either replacing or silencing disease-causing genes, potentially leading to complete recovery. In this review, we primarily focus on the latest applications of gene editing research in RP. We delve into the most prevalent genes associated with RP and discuss advancements in genome-editing strategies currently employed to correct various disease-causing mutations.

Targeted genome editing restores auditory function in adult mice with progressive hearing loss caused by a human microRNA mutation.

Mutations in microRNA-96 (MIR96) cause autosomal dominant deafness-50 (DFNA50), a form of delayed-onset hearing loss. Genome editing has shown efficacy in hearing recovery through intervention in neonatal mice, yet editing in the adult inner ear is necessary for clinical applications, which has not been done. Here, we developed a genome editing therapy for the MIR96 mutation 14C>A by screening different CRISPR systems and optimizing Cas9 expression and the sgRNA scaffold for efficient and specific mutation editing. AAV delivery of the KKH variant of Staphylococcus aureus Cas9 (SaCas9-KKH) and sgRNA to the cochleae of presymptomatic (3-week-old) and symptomatic (6-week-old) adult Mir9614C>A/+ mutant mice improved hearing long term, with efficacy increased by injection at a younger age. Adult inner ear delivery resulted in transient Cas9 expression without evidence of AAV genomic integration, indicating the good safety profile of our in vivo genome editing strategy. We developed a dual-AAV system, including an AAV-sgmiR96-master carrying sgRNAs against all known human MIR96 mutations. Because mouse and human MIR96 sequences share 100% homology, our approach and sgRNA selection for efficient and specific hair cell editing for long-term hearing recovery lay the foundation for the development of treatment for patients with DFNA50 caused by MIR96 mutations.

Safety and efficacy studies of CRISPR-Cas9 treatment of sickle cell disease highlights disease-specific responses

Fetal hemoglobin (HbF) reactivation expression through CRISPR/Cas9 is a promising strategy for the treatment of sickle cell disease (SCD). Here, we describe a genome editing strategy leading to reactivation of HbF expression by targeting the binding sites (BSs) for the LRF repressor in the γ-globin promoters. CRISPR/Cas9 treatment in healthy donor (HD) and patient-derived HSPCs resulted in a high frequency of LRF BS disruption and potent HbF synthesis in their erythroid progeny. LRF BS disruption did not impair HSPC engraftment and differentiation, but was more efficient in SCD than in HD cells. However, SCD HSPCs showed a reduced engraftment and a myeloid bias compared to HD cells. We detected off-target activity and chromosomal rearrangements, particularly in SCD samples (likely because of the higher overall editing efficiency), but did not impact the target gene expression and HSPC engraftment and differentiation. Transcriptomic analyses showed that the editing procedure results in the upregulation of genes involved in DNA damage and inflammatory responses, which was more evident in SCD HSPCs. This study provides evidences of efficacy and safety for an editing strategy based on HbF reactivation and highlights the need of performing safety studies in clinically relevant conditions, i.e., in patient-derived HSPCs.

Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro.

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70-Ku80 heterodimer (Ku), X-ray repair cross complementing 4 (XRCC4) in complex with DNA ligase 4 (X4L4) and XRCC4-like factor (XLF) form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were recently obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at residue resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs lead to the formation of XLF and X4L4 condensates in vitro, which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome-editing strategies.

Dual auxotrophy coupled red labeling strategy for efficient genome editing in Saccharomyces cerevisiae

The homologous recombination strategy has a long history of editing Saccharomyces cerevisiae target genes. The application of CRISPR/Cas9 strategy to editing target genes in S. cerevisiae has also received a lot of attention in recent years. All findings seem to indicate that editing relevant target genes in S. cerevisiae is an extremely easy event. In this study, we systematically analyzed the advantages and disadvantages of homologous recombination (HR) strategy, CRISPR/Cas9 strategy, and CRISPR/Cas9 combined homology-mediated repair (CRISPR/Case9-HDR) strategy in knocking out BY4742 ade2. Our data showed that when the ade2 was knocked out by HR strategy, a large number of clones appeared to be off-target, and 10 %–80 % of the so-called knockout clones obtained were heteroclones. When the CRISPR/Cas9 strategy was applied, 60% of clones were off-target and the rest were all heteroclones. Interestingly, most of the cells were edited successfully, but at least 60 % of the clones were heteroclones, when the CRISPR/Cas9-HDR strategy was employed. Our results clearly showed that the emergence of heteroclone seems inevitable regardless of the strategies used for editing BY4742 ade2. Given the characteristics of BY4742 defective in ade2 showing red on the YPD plate, we attempted to build an efficient yeast gene editing strategy, in which the CRISPR/Cas9 combines homology-mediated repair template carrying an ade2 expression cassette, BY4742(ade2Δ0) as the start strain. We used this strategy to successfully achieve 100 % knockout efficiency of trp1, indicating that technical challenges of how to easily screen out pure knockout clones without color phenotype have been solved. Our data showed in this study not only establishes an efficient yeast gene knockout strategy with dual auxotrophy coupled red labeling but also provides new ideas and references for the knockout of target genes in the monokaryotic mycelium of macrofungi.

Enhancement of erythropoietic output by Cas9-mediated insertion of a natural variant in haematopoietic stem and progenitor cells.

Some gene polymorphisms can lead to monogenic diseases, whereas other polymorphisms may confer beneficial traits. A well-characterized example is congenital erythrocytosis-the non-pathogenic hyper-production of red blood cells-that is caused by a truncated erythropoietin receptor. Here we show that Cas9-mediated genome editing in CD34+ human haematopoietic stem and progenitor cells (HSPCs) can recreate the truncated form of the erythropoietin receptor, leading to substantial increases in erythropoietic output. We also show that combining the expression of the cDNA of a truncated erythropoietin receptor with a previously reported genome-editing strategy to fully replace the HBA1 gene with an HBB transgene in HSPCs (to restore normal haemoglobin production in cells with a β-thalassaemia phenotype) gives the edited HSPCs and the healthy red blood cell phenotype a proliferative advantage. Combining knowledge of human genetics with precise genome editing to insert natural human variants into therapeutic cells may facilitate safer and more effective genome-editing therapies for patients with genetic diseases.

Efficient CRISPR-Cas12f1-Mediated Multiplex Bacterial Genome Editing via Low-Temperature Recovery.

CRISPR-Cas system is being used as a powerful genome editing tool with developments focused on enhancing its efficiency and accuracy. Recently, the miniature CRISPR-Cas12f1 system, which is small enough to be easily loaded onto various vectors for cellular delivery, has gained attention. In this study, we explored the influence of temperature conditions on multiplex genome editing using CRISPR-Cas12f1 in an Escherichia coli model. It was revealed that when two distinct targets in the genome are edited simultaneously, the editing efficiency can be enhanced by allowing cells to recover at a reduced temperature during the editing process. Additionally, employing 3'-end truncated sgRNAs facilitated the simultaneous single-nucleotide level editing of three targets. Our results underscore the potential of optimizing recovery temperature and sgRNA design protocols in developing more effective and precise strategies for multiplex genome editing across various organisms.

Melanin-based duplex Cas9 ribonucleoprotein nanomedicine for synergistic phototherapy and immunotherapy

Immune checkpoint blockade (ICB) has shown significant progress in treating triple negative breast cancer (TNBC). However, the standalone effectiveness of ICB is modest, offering patients only slight benefits. In this report, we utilize a hypoxia-responsive duplex genome editing strategy to target tumor cell programmed cell death ligand 1 (PD-L1) and protein tyrosine phosphatase non-receptor type 2 (PTPN2), which is synergized with photothermal therapy (PTT) to amplify the therapeutic effect of TNBC. In our approach, the melanin nanoparticles (MNP) serve as the delivery platform for transporting the CRISPR-CasPTPN2/PD-L1 (Cas-TD) system, which is cross-linked by a hypoxic-sensitive Azo linker and then further modified with tumor homing phenylboronic acid (PBA), resulting in a stimuli-responsive multifunctionality nanotheranostic platform (MPA@Cas-TD@PP). Aided by the guidance of PBA, nanoparticles selectively accumulate at tumor and then disintegrate with enhanced intracellular penetration. Importantly, PTT initiates the immunogenic cell death (ICD), turning the “cold” tumor into a “hot” one, laying the foundation for subsequent ICB. Inhibiting PTPN2 and PD-L1 can synergistically sensitize tumors to immunotherapy, which exhibit magnified immunotherapy effects by increasing activated cytotoxic CD8+ T cells and enhancing interferon-γ (IFN-γ)-mediated effects. In summary, this multifunctional nanotherapeutic platform provides a promising treatment approach for synergistic phototherapy and immunotherapy in TNBC.

Abstract 6323: Preclinical evaluation of CB-012, an allogeneic anti-CLL-1 CAR-T cell therapy, that exhibits specific and potent toxicity in acute myeloid leukemia (AML) xenograft models

Abstract Background: CLL-1 is a compelling therapeutic target for AML as it is highly expressed on AML tumor cells and leukemic stem cells but is not expressed on hematopoietic stem cells. CB-012 was engineered with a next-generation Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology and leverages both checkpoint disruption and immune cloaking armoring strategies to potentially improve antitumor activity. The CB-012 anti-CLL-1 CAR was developed with a fully human scFv and the CD28 costimulatory domain and is currently in development for the treatment of relapsed or refractory AML (r/r AML). Here we describe preclinical studies that supported the CB-012 IND clearance by the FDA in October 2023. Methods: Cas12a chRDNA guides were implemented to generate five genome edits in the manufacture of CB-012. A multiplex genome-editing strategy was designed to enhance the antitumor activity of CB-012 through prevention of GvHD, PD-1 checkpoint disruption, and suppression of allograft rejection. In vitro and in vivo studies evaluated the specificity of antigen binding, antigen-dependent activity, and toxicologic potential. Results: CB-012 demonstrated potent antigen-dependent expansion and cytotoxic activity against CLL-1+ human AML cell lines and patient-derived cells in co-cultures. In AML xenograft models, a single dose of CB-012 CAR-T cells resulted in robust tumor control, leading to significant prolongation of survival. CB-012 co-culture with multiple CLL-1-negative cell types representing vital tissues demonstrated that the anti-CLL-1 scFv does not exhibit tissue cross-reactivity. In an unbiased cell surface protein microarray, the anti-CLL-1 scFv demonstrated highly specific interaction with human CLL-1, with no detectable non-specific interactions. CB-012 CAR-T cells exhibited limited tissue infiltration and expansion in treatment naïve, immunocompromised murine models. Conclusion: CB-012, the first allogeneic anti-CLL-1 CAR-T cell therapy using both checkpoint disruption and immune cloaking armoring, demonstrated specific and potent CLL-1-targeted cytolytic activity in vitro and in vivo. Specificity of the anti-CLL-1 scFv was demonstrated in an unbiased protein-binding study and no adverse safety signals were observed from CB-012 in murine toxicology models. These preclinical studies supported the IND clearance of CB-012, which is being evaluated in the AMpLify trial, a Phase 1, first-in-human clinical trial for patients with r/r AML (NCT06128044). Citation Format: Brian Francica, Elizabeth Garner, Sai Namburi, Cian Colgan, Tristan Fowler, Devin Mutha, Art Aviles, Morena Stanaway, Raymond Guo, Zili An, Erin Kelly, Emilie Degagne, George Kwong, Leslie Edwards, Emma Jakes, McKay Shaw, Benjamin Schilling, Jeremy Huynh, Ricky Luu, Max Sidorov, Rhonda Mousali, Mikk Otsmaa, Peter Lauer, Justin Skoble, Steven Kanner. Preclinical evaluation of CB-012, an allogeneic anti-CLL-1 CAR-T cell therapy, that exhibits specific and potent toxicity in acute myeloid leukemia (AML) xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6323.

Nuclease-free precise genome editing corrects MECP2 mutations associated with Rett syndrome.

Rett syndrome is an acquired progressive neurodevelopmental disorder caused by de novo mutations in the X-linked MECP2 gene which encodes a pleiotropic protein that functions as a global transcriptional regulator and a chromatin modifier. Rett syndrome predominantly affects heterozygous females while affected male hemizygotes rarely survive. Gene therapy of Rett syndrome has proven challenging due to a requirement for stringent regulation of expression with either over- or under-expression being toxic. Ectopic expression of MECP2 in conjunction with regulatory miRNA target sequences has achieved some success, but the durability of this approach remains unknown. Here we evaluated a nuclease-free homologous recombination (HR)-based genome editing strategy to correct mutations in the MECP2 gene. The stem cell-derived AAVHSCs have previously been shown to mediate seamless and precise HR-based genome editing. We tested the ability of HR-based genome editing to correct pathogenic mutations in Exons 3 and 4 of the MECP2 gene and restore the wild type sequence while preserving all native genomic regulatory elements associated with MECP2 expression, thus potentially addressing a significant issue in gene therapy for Rett syndrome. Moreover, since the mutations are edited directly at the level of the genome, the corrections are expected to be durable with progeny cells inheriting the edited gene. The AAVHSC MECP2 editing vector was designed to be fully homologous to the target MECP2 region and to insert a promoterless Venus reporter at the end of Exon 4. Evaluation of AAVHSC editing in a panel of Rett cell lines bearing mutations in Exons 3 and 4 demonstrated successful correction and rescue of expression of the edited MECP2 gene. Sequence analysis of edited Rett cells revealed successful and accurate correction of mutations in both Exons 3 and 4 and permitted mapping of HR crossover events. Successful correction was observed only when the mutations were flanked at both the 5' and 3' ends by crossover events, but not when both crossovers occurred either exclusively upstream or downstream of the mutation. Importantly, we concluded that pathogenic mutations were successfully corrected in every Rett line analyzed, demonstrating the therapeutic potential of HR-based genome editing.

Genetic dissection of mutagenic repair and T-DNA capture at CRISPR-induced DNA breaks in Arabidopsis thaliana.

A practical and powerful approach for genome editing in plants is delivery of CRISPR reagents via Agrobacterium tumefaciens transformation. The double-strand break (DSB)-inducing enzyme is expressed from a transferred segment of bacterial DNA, the T-DNA, which upon transformation integrates at random locations into the host genome or is captured at the self-inflicted DSB site. To develop efficient strategies for precise genome editing, it is thus important to define the mechanisms that repair CRISPR-induced DSBs, as well as those that govern random and targeted integration of T-DNA. In this study, we present a detailed and comprehensive genetic analysis of Cas9-induced DSB repair and T-DNA capture in the model plant Arabidopsis thaliana. We found that classical nonhomologous end joining (cNHEJ) and polymerase theta-mediated end joining (TMEJ) are both, and in part redundantly, acting on CRISPR-induced DSBs to produce very different mutational outcomes. We used newly developed CISGUIDE technology to establish that 8% of mutant alleles have captured T-DNA at the induced break site. In addition, we find T-DNA shards within genomic DSB repair sites indicative of frequent temporary interactions during TMEJ. Analysis of thousands of plant genome-T-DNA junctions, followed up by genetic dissection, further reveals that TMEJ is responsible for attaching the 3' end of T-DNA to a CRISPR-induced DSB, while the 5' end can be attached via TMEJ as well as cNHEJ. By identifying the mechanisms that act to connect recombinogenic ends of DNA molecules at chromosomal breaks, and quantifying their contributions, our study supports the development of tailor-made strategies toward predictable engineering of crop plants.

An alpha-helical lid guides the target DNA toward catalysis in CRISPR-Cas12a

CRISPR-Cas12a is a powerful RNA-guided genome-editing system that generates double-strand DNA breaks using its single RuvC nuclease domain by a sequential mechanism in which initial cleavage of the non-target strand is followed by target strand cleavage. How the spatially distant DNA target strand traverses toward the RuvC catalytic core is presently not understood. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an α-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the DNA target strand by anchoring the crRNA:target strand duplex and guiding the target strand toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the REC2 domain pushes the crRNA:target strand duplex toward the core of the enzyme, while the Nuc domain aids the bending and accommodation of the target strand within the RuvC core by bending inward. Understanding of this critical process underlying Cas12a activity will enrich fundamental knowledge and facilitate further engineering strategies for genome editing.

When an Intruder Comes Home: GM and GE Strategies to Combat Virus Infection in Plants

Viruses are silent enemies that intrude and take control of the plant cell’s machinery for their own multiplication. Infection by viruses and the resulting damage is still a major challenge in the agriculture sector. Plants have the capability to fight back, but the ability of viruses to mutate at a fast rate helps them to evade the host’s response. Therefore, classical approaches for introgressing resistance genes by breeding have obtained limited success in counteracting the virus menace. Genetic modification (GM)-based strategies have been successful in engineering artificial resistance in plants. Several different approaches based on pathogen-derived resistance, antisense constructs, hairpin RNAs, double-stranded RNA, etc., have been used to enhance plants’ resistance to viruses. Recently, genome editing (GE) strategies mainly involving the CRISPR/Cas-mediated modifications are being used for virus control. In this review, we discuss the developments and advancements in GM- and GE-based methods for tackling viral infection in plants.

Monitoring Genomic Structural Rearrangements Resulting from Gene Editing.

The cytogenomics-based methodology of directional genomic hybridization (dGH) enables the detection and quantification of a more comprehensive spectrum of genomic structural variants than any other approach currently available, and importantly, does so on a single-cell basis. Thus, dGH is well-suited for testing and/or validating new advancements in CRISPR-Cas9 gene editing systems. In addition to aberrations detected by traditional cytogenetic approaches, the strand specificity of dGH facilitates detection of otherwise cryptic intra-chromosomal rearrangements, specifically small inversions. As such, dGH represents a powerful, high-resolution approach for the quantitative monitoring of potentially detrimental genomic structural rearrangements resulting from exposure to agents that induce DNA double-strand breaks (DSBs), including restriction endonucleases and ionizing radiations. For intentional genome editing strategies, it is critical that any undesired effects of DSBs induced either by the editing system itself or by mis-repair with other endogenous DSBs are recognized and minimized. In this paper, we discuss the application of dGH for assessing gene editing-associated structural variants and the potential heterogeneity of such rearrangements among cells within an edited population, highlighting its relevance to personalized medicine strategies.

Recent advances in genome editing strategies for balancing growth and defence in sugarcane (Saccharum officinarum).

Sugarcane (Saccharum officinarum ) has gained more attention worldwide in recent decades because of its importance as a bioenergy resource and in producing table sugar. However, the production capabilities of conventional varieties are being challenged by the changing climates, which struggle to meet the escalating demands of the growing global population. Genome editing has emerged as a pivotal field that offers groundbreaking solutions in agriculture and beyond. It includes inserting, removing or replacing DNA in an organism's genome. Various approaches are employed to enhance crop yields and resilience in harsh climates. These techniques include zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/associated protein (CRISPR/Cas). Among these, CRISPR/Cas is one of the most promising and rapidly advancing fields. With the help of these techniques, several crops like rice (Oryza sativa ), tomato (Solanum lycopersicum ), maize (Zea mays ), barley (Hordeum vulgare ) and sugarcane have been improved to be resistant to viral diseases. This review describes recent advances in genome editing with a particular focus on sugarcane and focuses on the advantages and limitations of these approaches while also considering the regulatory and ethical implications across different countries. It also offers insights into future prospects and the application of these approaches in agriculture.

Metabolic engineering and genome editing strategies for enhanced lipid production in microalgae

Metabolic engineering and genome editing strategies for enhanced lipid production in microalgae

Emerging lipid–polymer hybrid nanoparticles for genome editing

Lipid–polymer hybrid nanoparticles are rapidly emerging as a major class of efficient delivery systems for biomedical applications. This review showcases and discusses the designs and major advances of lipid–polymer hybrids for genome editing strategies.